Leucovorin (LV), an equimolar racemic mixture of the diastereoisomers, 6-S- and 6-R-5CHO-H4PteGlu, must be given following the chemotherapeutic administration of high dose Methotrexate (MTX) to prevent death of the host. The mechanism for selective "rescue" of normal tissues, while achieving maximal cytotoxicity in tumor cells, is not well understood at the biochemical level. The biological activity of the two diastereoisomers of LV is also not well known. A new site of intracellular interaction between MTX and LV is proposed to be the enzyme, folyl polyglutamate synthetase (FPGS). In vitro murine liver FPGS enzyme assays have shown both isomers of LV to have substrate activity and to be polyglutamated. MTX is well known to be polyglutamated. The pattern of MTX-polyglutamates in cells in tissue culture and transplanted tumors in mice will be examined using high performance liquid chromatographic separation of polyglutamates. The cultures or the mice bearing tumors, will be dosed with 3H-MTX (control), or pulsed with 3H-MTX followed by "rescue" doses of leucovorin. The effect of leucovorin on MTX-polyglutamation will be examined comparing MTX-sensitive and MTX-resistant cells, and comparing normal tissues and malignant cells. Pharmacolokinetic studies of the LV diastereoisomers will also be performed. Both diastereoisomers will be synthesized and radiolabeled. Direct measurements of cellular uptake will be performed. Oral absorption and metabolism will be compared with the intravenous metabolism of LV diastereoisomers. Techniques for performing assays and synthesizing necessary compounds are presently available. All planned studies would reveal information basic to an understanding of the biological activity of Leucovorin and would examine a new intracellular site of folate-antifol interaction.